Zymogen activation peptides (ZAP) in the diagnosis of disease

ABSTRACT

Pancreatic disease can be diagnosed by assaying a patient&#39;s body fluid such as serum or urine, for pancreatic activation peptides (PAP) released from zymogens by proteolytic activation. Particularly useful are peptides having C-terminal D 4  K sequences. The method uses polyclonal or monoclonal antibodies generated and selected for C-terminal specificity on PAP so that the tests only report free PAP not parent zymogen. Also described are peptides and antibodies labelled with revealing agents and/or immobilised on solid supports and their use in diagnostic assays and kits. In pancreatic disease the tests distinguish necrotising from oedematous acute pancreatitis and permit severity prediction and monitoring as well as diagnosing chronic pancreatitis in exacerbation. Tests reporting free activation peptides from the zymogens prophospholipase A, procolipase and proelastases are also applicable in non-pancreatic diseases where activation of these zymogens, sharing sequence homology in the activation peptide C-terminal region with pancreatic isoenzymes, forms part of the molecular pathology of the condition.

This application is a continuation of application Ser. No. 145,857filed, Jan. 20, 1988 (now abandoned) which is a continuation-in-part ofour application Ser. No. 078737 filed Jul. 28th 1987 now U.S. Pat. No.4,948,723, which is itself a continuation-in-part of our applicationSer. No. 003728 filed Jan. 16th 1987, now abandoned.

The activation by limited proteolysis of precursor forms, or zymogens,of proteolytic and lipolytic enzymes, as well as protein co-factors,plays an important part in physiological and pathological processes. Thedetection and quantitation of these events by measuring the activeenzyme or co-factor formed, has several theoretical and practicaldisadvantages. A good example of this is seen in the pancreatitis groupof diseases described in detail below. The present Application is basedon a new approach to the detection and quantitation of precursoractivation by measuring levels of the released activation peptides usingspecific C-terminally directed anti-peptide antibodies which only bindto free peptides and not to parent precursor molecules.

Acute and chronic pancreatitis are human diseases which have shown asignificant increase in frequency in recent years [1]. Acutepancreatitis presents as an abdominal emergency, whereas chronicpancreatitis in general involves the differential diagnosis of chronicor intermittent abdominal pain.

The exocrine pancreas produces and stores a range of digestive enzymes,including amylase and lipase, biosynthesised in active form, and othersincluding trypsinogens, chymotrypsinogens, proelastases,procarboxypeptidases, and prophospholipases A2, synthesised as inactiveproenzymes or zymogens. Zymogen activation normally occurs followingsecretion of pancreatic juice into the duodenal lumen, and involves thespecific catalytic conversion of trypsinogen to active trypsin byduodenal enteropeptidase (E.C. 3.4.21.9). This removes from trypsinogenan amino-terminal oligopeptide containing the sequencetetra-L-aspartyl-L-lysine, an event followed by tryptic activation ofthe other proenzymes in a cascade of proteolytic cleavage. Activationpeptides of pancreatic zymogens released in the course of thisphysiological process are thought to be degraded by duodenaloligopeptidases.

Acute pancreatitis is characterised by two distinct clinicopathologicaltypes [2]. In acute oedematous pancreatitis, pancreatic acinar celldamage is accompanied by leakage of inactive digestive zymogens withactive amylase and lipase into the peritoneal cavity and circulation.The active enzymes are identifiable in the resulting ascitic fluid aswell as in blood and urine, while proteinase digestive zymogens andprophospholipase remain unactivated. Although local inflammation and fatnecroses due to active lipase occur, the condition is generallynon-lethal and recovers within a few days of conservative management. Inacute nectorising pancreatitis on the other hand, pancreatic acinar celldamage and digestive enzyme release is associated with varying degreesof digestive zymogen activation. Although complexes form betweenpancreatic proteinases and circulating macromolecular inhibitors, someof these complexes remain catalytically active. Phospholipase A₂ is alsoactive in plasma. Widespread local and disseminated multiorgan damageresults in a high morbidity and mortality.

The biochemical diagnosis of acute pancreatitis has traditionally reliedon the detection in plasma, serum, urine, or ascitic fluid, ofpancreatic digestive enzymes or their zymogens released from thepancreas itself as a result of the disease. The quantitation of totalamylase activity in serum has been the principle diagnostic testemployed [reviewed in ref. 3]. The arrival of sensitive immunologicalmethods permitted the development of assays for pancreatic proteolyticenzymes and their zymogens including trypsin and trypsinogens [4-18],elastase [19,20], chymotrypsin [21], phospholipase [22,23],carboxypeptidases [24] and, in addition, lipase [25,26] and pancreatictrypsin inhibitor [27,28]. Some of these immunoassays particularly thoseusing polyclonal antisera were complicated by the variable proteolyticdegradation of their target molecules. Additional problems with theseassays arise due to steric inhibition of antibody binding to targetenzymes in complexes with macromolecular inhibitors [29,30]. Furthermoreantibody recognising both parent zymogen and active enzyme is unable todistinguish between the two.

Amylase assays were improved to differentiate between pancreaticisoamylase and amylase activity contributed by other organs,particularly salivary glands [31-33]. Simultaneous detailed assessmentof many of these tests in multiple patients with acute pancreatitis has,however, shown that the detection of lipase or immunoreactivetrypsinogen does not improve initial diagnostic accuracy over themeasurement of pancreatic isoamylase alone, and does not permit thebiochemical recognition of the severe acute necrotising disease [34].Amylase estimation in the diagnosis of pancreatic disease is complicatedby sporadic cases of hyperamylasaemia, the need to distinguishpancreatic from other sources of amylase, the variable degradation ofamylase activity in association with severe acute pancreatitis, and theinability to equate serum amylase levels with the severity ofpancreatitis or to identify the onset of necrosis. Serum amylaseestimations may also be unreliable in the recognition of ethanol-inducedpancreatitis [35-37]. Attempts to improve the accuracy of severityprediction by assay for catalytically active pancreatic phospholipase A2may prove a more fruitful approach [38]. This however is likely to havedifficulties with specificity since phospholipases A2 are present inmacrophages, granulocytes, platelets and other cells [39] and areelevated in blood in other acute abdominal conditions and septic shock[40].

The principal clinical diagnostic difficulty in acute pancreatitistherefore has been to distinguish severe abdominal pain withhyperamylasemia due to oedematous pancreatitis, from the more seriousnecrotising form in the earliest hours of the disease when intensivetreatment, the use of specific enzyme inhibitors, and even surgicalintervention and subtotal resection, may be life saving.

In chronic pancreatitis the diagnostic problem is different sincesubacute or intermittent abdominal pain due to chronic pancreatitis maynot be associated with hyperamylasemia and distinction is required fromthe many other potential causes of such symptoms.

THE INVENTION

The present invention is based upon the specific assay in body fluidsfor free pancreatic activation peptides PAP (the activation peptides ofpancreatic zymogens specifically cleaved by limited proteolysis duringactivation) having available carboxy-termini. Such assays will, for thefirst time, provide a precise method for recognising and quantifyingzymogen activation. Our invention is based on our appreciation thatdifferent forms of pancreatitis are associated with the specific releaseor otherwise, of PAP. This general method will distinguish between formsof pancreatitis occurring with or without zymogen activation and will beclinically applicable to the diagnosis, severity prediction, anddisease-course-monitoring in acute pancreatitis. It will also provide aprecise diagnostic test for chronic pancreatitis in exacerbation. SincePAP sequences may be found in some other cells and tissues, assays forPAP may be useful diagnostically in other conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the effect of various calcium concentrations on bindingto ¹²⁵ I-labeled YD₄ K in sera from three rabbits.

FIG. 2a and FIG. 2b show elution profiles of anti-D₄ K anitsera fromrabbit 2 and rabbit 17, respectively, after affinity chromatography onimmobilized D₄ K, showing the presence of Ca²⁺ -dependent and Ca²⁺-independent antibodies.

FIG. 3 demonstrates displacement by synthetic peptides of ¹²⁵ I-labeledYD₄ K bound to specific antisera.

FIG. 4 shows displacement of binding by ¹²⁵ I-YD₄ K by trypsinogen(purified and in pancreatic juice) with and without enterokinaseactivation.

FIG. 5a and FIG. 5b depict the elution profile of frog skin secretionfrom D-8 reverse phase column and the immunoreactivity in C-8 fractions,respectively.

FIG. 6 shows the results of radiometric assay-type 1 wherein wells werecoated with 60 μg/ml solution of YD₄ K-BSA and tested with Ca²⁺-independent antibody at a working dilution of 1:50. Displacement withD₄ K and detection with ¹²⁵ I-goat anti-rabbit antibody is shown.

FIG. 7 depicts the results of radiometric assay-type 2 wherein wellswere coated with 50 μg/ml solution of Ca²⁺ -independent antibodies.Competitive displacement of ¹²⁵ I-YD₄ K with D₄ K is shown.

FIG. 8 demonstrates the generation of D₄ K from dog pancreatic slices byenterokinase activation of trypsinogen.

FIG. 9 shows the clearance from serum of a single intravenous dose ofYD₄ K in a 30 kg greyhound.

FIG. 10 depicts the elution profile on sepharose G15 and theconcentration of D₄ K in samples of JS sera.

FIG. 11 shows the results of a TAP assay performed on pancreatic sera.

FIG. 12a, FIG. 12b and FIG. 12c demonstrate serial serum and urinelevels of D₄ K in patients TP, CM and CTM, respectively, presenting withacute pancreatitis.

DETAILED DESCRIPTION

In the test, a sample of the patient's body fluid, particularly bloodplasma, serum, urine or ascites will be assayed for one or more PAP.Cerebrospinal fluid may also be assayed. PAP available for such astrategy include the tetra-L-aspartyl-L-lysine (D₄ K in the singleletter amino acid code used in relation to this and subsequentlymentioned PAP) containing trypsinogen activation peptides (TAP) in whichthe D₄ K sequence is itself resistant to pancreatic proteolysis. Othersuitable activation peptides are DSGISPR [41] the activation peptide ofhuman pancreatic prophospholipase A2 (PLAP) and the APGPR [42]activation peptide of procolipase (CLAP). In addition proelastase I andII activation peptides (PEAP) [43] or derivatives of their limitedproteolysis, as well as the large (90-100 amino acid) amino terminalmoiety cleaved from procarboxypeptidases A and B, may also beconsidered. Of particular importance in this invention is the strategyof using antibody specifically carboxy-terminally directed against PAP.This will ensure that antibody binding and a positive result in theassay will only be reported when zymogen activation by limitedproteolysis has indeed occurred, and that zymogens themselves in whichPAP are linked by their carboxy-termini, will not be recognised.

Although the above example addresses the invention to distinguishing andcharacterising different forms of pancreatitis, the principle applies toother diseases. These include PLAP assays to assess activation ofcellular phospholipases A particularly from activated macrophagesinvolved in the liberation of pro-inflammatory mediators in diseasessuch as rheumatoid arthritis, Crohn's disease and dermatomyositis. Theinvolvement of lysolecithin and phospholipases A in demyelinatingdisorders indicates the further relevance of PLAP assays to theseconditions. Further considerations in respect of PLAP assays inadditional acute disorders and of assays for CLAP and PEAP are givenlater in this document under the heading "Other PAP Assays of theInvention".

EXAMPLES Immunoassay of Trypsinogen Activation Peptides (TAP)

In the following description, reference is frequently made to theimmunoassay of trypsinogen activation peptides TAP. This is but oneexample of PAP and what is described in the following description inrelation to TAP applies to the other PAP described above and below. Whenreference is made to TAP this is not to imply that the TAP that will bepresent will necessarily only be the pentapeptide D₄ K itself sinceadditional amino terminal amino acid residues may be present. Thesediffer among trypsinogen isoenzymes or may have been modified by aminoterminal degradation. The TAP peptides recognised in the assay willinclude the D₄ K sequence at the carboxyterminus and it is the D₄ Ksequence that is to be recognised.

The activation peptides of trypsinogens are highly conserved invertebrate evolution and contain the polyanionic sequence D₄ K upstreamof the lysyl-isoleucyl target bond that is cleaved during specificproteolytic activation [44,45]. The assay of the sample for the aminoacid sequence D₄ K is most satisfactorily carried out using an antibodythat recognises the D₄ K sequence with specificity for C-terminal D₄ Kpeptides and such antibodies form a further aspect of the presentinvention. These antibodies may be polyclonal or monoclonal. Thepolyclonal antibodies of the invention can be raised in animals byconventional techniques using as the immunogen D₄ K or D₄ K having ashort leader sequence, e.g. alanyl-propyl-phenylalanyl with or withoutcysteine at the amino terminus and either as free D₄ K containingpeptide or D₄ K peptide haptenised prior to use as an immunogen. Wherehaptenisation is required, the D₄ K sequence or peptides including itcan be chemically bonded to conventional peptide haptens such as bovineserum albumin (BSA) or thyroglobulin (TG).

When monoclonal antibodies are required, they may be prepared byconventional hybridoma technology, again using D₄ K or an amino acidsequence including D₄ K or D₄ K haptenised prior to use as theimmunogen. Monoclonal antibodies to D₄ K may also be generated by usingD₄ K bound to previously prepared anti-D₄ K antibody as the immunogen.

The present invention is based upon our appreciation that assay of PAPsuch as the D₄ K sequence can provide valuable unambiguous informationconcerning the existence and nature of pancreatic disorders in apatient. The exact way in which the assay is carried out is not criticaland use may be made of any of the available direct or indirect(competitive) assays as well as two-site sandwich assays and assaysinvolving quantification of antibody occupancy. The assay will normallyinvolve the formation, in samples where D₄ K peptides are present, of aconjugate between the peptide including the D₄ K sequence and theantibody, said conjugate carrying a revealing label and being formedeither in the solid phase or in the liquid phase of a solid/liquid phasereaction mixture, separating the solid phase from the liquid phase anddetermining the presence of or amount of the revealing label in eitherthe solid phase or the liquid phase as a measure of the presence of oramount of, respectively, peptide including the D₄ K sequence in thesample. Competitive assays normally require the attachment of therevealing label to competing D₄ K but labelling of a second anti-D₄ Kantibody is necessary for the two-site sandwich assay. Any of theconventional revealing labels can be used, enzyme labels or radioactivelabels being preferred although immunofluorescence can also be used. Theselection of the enzyme or radioactive label may be ¹²⁵ I, one of thepreferred radioactive labels, while horseradish peroxidase or alkalinephosphatase are the preferred enzymes. Biotin may also be used.Polyclonal or monoclonal anti-D₄ K carrying a revealing label form afurther aspect of the present invention as does the pentapeptide D₄ Khaving a revealing label directly attached to one of its constituentamino acids and polypeptides including the labelled pentapeptideconjugated to another polypeptide. Amplification methods may be employedsuch as those which link alkaline phosphatase coupled to D₄ K peptideinto the conversion of NADP⁺ to NAD⁺. The NAD⁺ so formed thencatalytically activates an NAD⁺ -specific redox cycle yielding anintensely coloured formazan dye [46].

The anti-D₄ K can be used, in accordance with the present invention, inconventional radio-immunoassays, in enzyme-linked or enzyme-multipliedimmunoassays, in accordance with developed techniques, or in alternativeor homogeneous immunoassays employing chemiluminescence,bioluminescence, photon emission fluorometry, electroluminescence,polarisation fluorescence, time resolved or other techniques. Thesewould form the basis of reference laboratory immunoassays or simplifiedmethods applicable to use in satellite laboratory, physicians office,emergency department, ward or intensive care unit. These methodsfrequently require the anti-D₄ K antibody or D₄ K itself to beimmobilised by attachment to an inert solid substrate, such as latexparticles or polystyrene balls or powder which can be packed into acolumn through which the test solutions can be run and such immobilisedpolyclonal or monoclonal anti-D₄ K and immobilised peptides comprisingthe D₄ K sequence having the lysine (K) as the carboxy terminus, with orwithout the attachment of a revealing label, form further aspects of theinvention.

The invention also provides diagnostic test kits. Such kits comprise, asone component a labelled peptide of the invention (alone or conjugatedto another peptide) or a labelled antibody of the invention.Alternatively, the test kit is one comprising a solid and a liquidcomponent where the solid component is an immobilised peptide orantibody of the invention and one of the components is labelled,preferably with an enzyme or radioactive label.

In general, assays in accordance with the present invention usingimmobilised anti-D₄ K will include the step of bringing a liquid sample,suspected of including D₄ K peptides, into contact with a solid phaseincluding a polyclonal or monoclonal anti-D₄ K in competition with D₄ Kcarrying a revealing agent, and assaying either the solid phase or theliquid phase for the presence of D₄ K carrying the revealing agent andtaking the presence of that conjugate as a measure of the D₄ K contentof the sample. Assays in accordance with the present invention will alsoinclude the method of bringing a liquid sample suspected of including D₄K peptides, into contact with a solid phase with attached polyclonal ormonoclonal anti-D₄ K, separating the solid from the liquid phase, andquantifying the D₄ K bound by immobilised anti-D₄ K, using a monoclonalsecond anti-D₄ K antibody carrying a revealing agent. An alternativemethod may also be used of quantifying antibody or immobilised D₄ Kpeptide occupancy in one or two dimensions on solid phase after exposureto the test sample, by the subsequent use of D₄ K peptides or antibodycoupled to a revealing agent. Assays in accordance with the presentinvention will also include the method of adding anti-D₄ K polyclonal ormonoclonal antibody with or without revealing agent, to the sample to betested and bringing the treated sample into contact with D₄ K peptidesimmobilised on solid phase either directly or via an intermediate andsuch immobilised D₄ K peptide forms part of the present invention. D₄ Kpeptides on membranes or other solid phase support may be in the form ofa disc which may have a central well, ring, or minicolumn. The test willbe read by the intensity and/or distribution of anti-D₄ K binding to theimmobilised D₄ K using the attached revealing agent or a revealing agentcoupled to a second antibody to anti-D₄ K Ig. Assays in accordance withthe present invention will also include the method of bringing thesample suspected of including D₄ K peptides into contact with a complexcomprising anti-D₄ K antibody and D₄ K, immobilised on an inert solidsupport and with or without a revealing agent. If the D₄ K is directlybound to the support, the antibody carries any revealing agent and viceversa. The displacement of initial D₄ K from the solid phase by D₄ Kpeptides in the sample may then be quantified.

The assay of the present invention may also be used to monitor theseverity progress of pancreatitis. To do this, samples of body fluid areremoved from the patient on at least two separate occasions spaced apartfrom one another by one half to four hours or longer and each sample isassayed for the concentration of D₄ K peptide. By taking several samplesfrom the patient over a 24 hour period, it is possible to determinewhether the severity of the patient's condition is changing and is suchas to require surgery or other specific treatment.

The D₄ K peptides and other PAP are also of value in that they can beused for the purification, by affinity chromatography, of polyclonal ormonoclonal anti-D₄ K or other anti-PAP. In such purification procedures,the D₄ K peptide or other PAP will normally be immobilised in asterically accessible manner, on a solid support such as polystyrene ora polysaccharide such as a polydextran, a liquid sample containingrelatively impure anti-D₄ K or other anti-PAP brought into contact withthe immobilised D₄ K peptide or other PAP forming a D₄ K/anti-D₄ K orother PAP/anti-PAP conjugate on the support. After washing the solidphase the anti-D₄ K or other anti-PAP is eluted to give a solutioncontaining a relatively more pure form of anti-D₄ K or other anti-PAP.

The following Examples are given to illustrate the present invention.

In these Examples, single letter and three letter amino acidabbreviations are used as follows:

    ______________________________________                                        D            Asp         Aspartic Acid                                        K            Lys         Lysine                                               Y            Tyr         Tyrosine                                             C            Cys         Cysteine                                             A            Ala         Alanine                                              P            Pro         Proline                                              F            Phe         Phenylalanine                                        S            Ser         Serine                                               G            Gly         Glycine                                              I            Ile         Isoleucine                                           R            Arg         Arginine                                             T            Thr         Threonine                                            ______________________________________                                    

SYNTHESIS OF ASP₄ -LYS (D₄ K) PEPTIDES

Peptides corresponding to the activation peptides found in humantrypsinogens, Asp-Asp-Asp-Asp-Lys (D₄ K), andAla-Pro-Phe-Asp-Asp-Asp-Asp-Lys (APFD₄ K), were synthesised with extraresidues Tyr and Cys on their respective N-termini to provideside-chains that could be chemically cross-linked to protein carriers.Since the D₄ K sequence is degraded by aggressive acids, the base labileN-fluorenylmethoxycarbonyl (Fmoc) group was used for reversiblealpha-N-terminal protection [47]. The peptides YD₄ K and CAPFD₄ K wereassembled on polystyrene solid phase supports with the acid-labilep-alkoxybenzyl alcohol C-terminal linker [48]. A modification of theprocedure of Meienhofer et al [49] was used for the synthesis, omittingthe dioxane:H₂ O wash which was found to be unnecessary, anddeprotecting with 20% piperidine in DMF rather than dichloromethane.

Our initial studies showed that if one protected aspartyl group wascoupled to the immobilised C-terminal (N-t-Boc)Lys residue, thedipeptide Asp-Lys cyclised to a diketopiperazine on base-catalysedremoval of the alpha-amino Fmoc group, and the moiety was released fromthe resin. The tetra-aspartyl sequence was therefore assembled usingprotected Asp-Asp dipeptides previously prepared, usinghydroxybenzotriazole coupling to suppress racemisation during couplingto the resin-bound peptide. Completion of peptide bond formation aftereach addition was monitored with ninhydrin, or fluorescamine forProline. Double couplings were required for each Asp-Asp dipeptide andfor Fmoc (Trt) Cys. After simultaneous cleavage from the resin anddeprotection of t-butyl side-chain protecting groups with 50% TFA in CH₂Cl₂ (containing also 5% ethyl methyl sulphide in the case of CAPFD₄ K),peptides were purified by successive gel filtration and ion-exchangechromatography to yield the required peptides in 62% (YD₄ K) and 35%(CAPFD₄ K) yields. The peptides were shown to be homogeneous as singleninhydrin-positive spots on high voltage paper electrophoresis at pH 6.5(R_(Asp) YD₄ K=0.79, and R_(Asp) CAPFD₄ K= 0.76), and satisfactory aminoacid analysis after acid hydrolysis. We have previously shown by ¹³C-NMR that trypsinogen activation peptides synthesised by this procedureare free of alpha-beta-transpeptidation [50].

HAPTENISATION OF D₄ K PEPTIDES

Two haptens were prepared by specific amino terminal coupling ofsynthetic trypsinogen activation peptides to protein carriers. YD₄ K wascross-linked to bovine serum albumin (BSA) by bis-diazotisation with theTyr residue using benzidine dihydrochloride as described by Bassiri andUtiger (1972) [51]. Amino acid analysis of the purified adduct BSA-YD₄ Kshowed a substitution of 8 peptides per BSA molecule. The sulphydrylgroup of the peptide CAPFD₄ K was coupled to epsilon-amino groups of Lyson bovine thyroglobulin (TG) using the heterobifunctional linkerm-moleimidobenzoyl-N-hydroxysuccinimide [52] to yield TG-CAPFD₄ K with asubstitution of 40 molecules of peptide per thyroglobulin molecule.

GENERATION OF SPECIFIC ANTI-D₄ K ANTISERA

Emulsions of equal amounts of Freund's complete adjuvant (MilesLaboratories) with BSA-YD₄ K 2.9 mg/ml or TG-CAPFD₄ K 1.1 mg/ml wereprepared by sonication on ice. NSW rabbits were immunised intradermallyand intramuscularly with 2.0 ml of one or other emulsion and boostedusing emulsions or corresponding hapten-peptide with Freund's incompleteadjuvant given each month. Sera were obtained before immunisation and 10days after each challenge and tested for the presence of anti-D₄ Kantibodies using a solid-phase immunoradiometric assay. BSA-YD₄ K andTG-CAPFD₄ K conjugates were immobilised in separate 96-wellpolyvinylchloride microtitre plates (Dynatech No. 1-220-24) byincubation of 50 ul at 60 μg/ml protein in 50mM Tris-HCL, 20mM CaCl₂,0.1% sodium azide (TCA-buffer) at 4° C. for 16 hours. Plates were thenwashed at room temperature with TCA-containing 10% (v/v) heat-irradiatedhorse serum (TCA-HS, Tissue Culture Services, Berkshire, UK), and excesssites blocked by incubation with TCA-HS for 1 hour. 50 μl aliquots ofimmune sera to be tested were then added to the wells in the presence orabsence of D₄ K 10⁻⁵ M and incubated for 2 hours at room temperature.Antisera from rabbits challenged with BSA-YD₄ K was tested onimmobilised TG-CAPFD₄ K and vice-versa. Plates were then washed x3 withTCA-HS and incubated for a further 16 hours with 50 μl ¹²⁵ I-goatantirabbit Ig (Miles AFA) 50,000 cpm per well in TCA-HS. Plates werewashed, dried, and the wells counted in an LKB gamma counter. Titre ofspecific anti-D₄ K antibody was obtained from the difference in cpmbound in the presence or absence of competing D₄ K peptide 10⁻⁵ M.

3 of 6 Rabbits immunised with BSA-YD₄ K developed anti-D₄ K antibodieswithin 3 weeks. The titre in these animals increased over 2 to 3 months,then declined. In two of these animals the titre rose again withcontinued challenge to peak at 4 and again at 6 months. Three of thefour rabbits immunised with TG-CAPFD₄ K responded with anti-D₄ Kantibodies with peaks in the specific antibody titre at 2 and 3 andagain at 6 months.

The presence of Ca²⁺ ion was found to enhance the binding of antibodiesin immune sera to activation peptides, the extent of enhancement beingdifferent in individual rabbits. The sera from one rabbit (1) exhibitedtotal dependence on Ca²⁺ binding to I-YD₄ K being reduced to backgroundlevel in the presence of chelating amounts of EDTA. In three rabbits(FIG. 1) binding increased in a concentration-dependent manner to amaximum observed at and above 1mM Ca²⁺ and thereafter fell up to 40mMCa²⁺. Other divalent metal ions tested Mg²⁺, Zn²⁺, Ba²⁺, Hg²⁺ did notincrease binding. The Ca²⁺ dependency of subpopulations of anti-D₄ Kantibodies was thought to be the result of the Ca²⁺ chelating propertiesof the two available pairs of Asp-beta carboxyls and the selectiverecognition of the peptide-Ca²⁺ chelate, rather than D₄ K peptide alone,by rabbit host immunocytes following spontaneous a Ca²⁺ binding in vivo.

AFFINITY PURIFICATION OF ANTI-D₄ K Ig FROM SPECIFIC ANTISERA Preparationof Affinity Adsorbent

Activated CH-Sepharose 4B (Pharmacia Fine Chemicals) was suspended in1mM HCl (100 ml), and washed with a further 900 ml of 1mM HCl. ThenTyr-Asp-Asp-Asp-Asp-Lys (60 rag) in 25 ml of 0.1M NaHCO₃ containing 0.5MNaCl was added and the gel shaken for 1 hour at 4° C. Excess ligand waswashed away with 100 ml of 0.1M NaHCO₃, and remaining active sitesblocked by shaking the gel with 0.1M Tris-HCl (pH 8) at 4° C. for 1hour. The gel was then washed with 50 ml 0.1M sodium acetate (pH 4)containing 0.5M NaCl, 50 ml 0.1M Tris-HCl (pH 8), then these were washedtwice more.

A small portion of the gel, hydrolysed in 6M HCl at 110° C. for 20 hour,and subjected to amino acid analysis, showed that the extent ofsubstitution was 1.10 mmols/ml of gel.

Affinity Chromatography on Immobilised YD₄ K

Two serum samples taken 6 weeks apart from rabbits, 1 and 2 (R₁ , R₂)immunised with BSA-YD₄ K, were pooled separately. Two similar samplesfrom R17 and R18 immunised with TG-CAPFD₄ K were pooled together. Thesepooled sera were subjected separately to affinity chromatography asfollows: equal volumes of saturated solutions of ammonium sulphate wereadded to the sera, and the mixture left overnight at 4° C. Theprecipitated proteins were collected by centrifugation at 9,200 xgav at4° C., and resuspended in the original volumes (see Table 1) of 50mMTris-HCl (pH 7.4), 150 mM NaCl, 20mM CaCl₂, and dialysed against two2-liter changes of the same buffer. The dialysed protein was applied toa 1.5×10 cm column of the Sepharose-immobilised D₄ K affinity matrix ata flow rate of 20 ml/h. Unbound protein was eluted with operationalbuffer until absorbance returned to zero. Then Ca²⁺ -dependentantibodies were displaced using 50mM Tris (pH 7.4), 150mM NaCl with 50mMEDTA followed by elution of Ca²⁺ -independent antibodies with 1Mpropionic acid. Fractions were dialysed, assayed for activity by binding¹²⁵ I-YD₄ K (specific radioactivity 2.7×10⁸ cpm/μg) and antibodycontaining fractions pooled.

The elution profile of immunoglobulins from rabbit 1 showed that a widepeak of antibodies was obtained by elution with EDTA, but no protein orantibody eluted in 1M propionic acid (FIG. 2a) confirming that anti-D₄ Kantibodies present in this serum were Ca²⁺ -dependent. Table 1 showsthat antisera from Rabbit 2 which had also been immunised with BSA-YD₄ Kcontained Ca²⁺ -dependent and Ca²⁺ -independent anti-D₄ K antibodies.This suggests the presence of two epitopes on the D₄ K molecule and thatthe recognition of one or both depended on the individual animal. Theelution profile of pooled sera from rabbits 17 and 18 immunised withTG-CAPFD₄ K (FIG. 2b) demonstrates both Ca²⁺ -dependent antibodiesdisplaced with EDTA and Ca²⁺ -independent antibodies displaced using 1Mpropionic acid.

                                      TABLE 1                                     __________________________________________________________________________    AFFINITY PURIFICATION ON YD.sub.4 K LINKED TO SEPHAROSE OF                    CALCIUM-DEPENDENT AND CALCIUM-INDEPENDENT ANTIBODIES                                           Vol (ml)                                                                           Total Protein (mg)                                                                       Total Activity                                                                         Specific Activity                                                                       Yield                                                                              Purification         __________________________________________________________________________    Rabbit 1 -                                                                    Imunized with BSA-YD.sub.4 K:                                                 Pooled serum      5   500        175.sup.a                                                                              0.35      100   1                   EDTA-peak         5   0.63       105.sup.a                                                                              66        60   180                  Propionic acid peak                                                                             8   0           0.sup.a 0         0    --                   Rabbit 2 -                                                                    Immunized with BSA-YD.sub.4 K:                                                Pooled serum     16   1092       286.sup.b                                                                              0.26      100   1                   EDTA-peak        11   3.3         48.sup.b                                                                              14.3      16.7 55                   Propionic acid peak                                                                            37   9.0        152.sup.b                                                                              17.0      53.2 65                   Rabbits 17/18 -                                                               Immunized with TG-CAPFD.sub.4 K:                                              Pooled serum     21   1848       709.sup.b                                                                              0.38      100   1                   EDTA-peak        52   9.5         90.sup.b                                                                              9.47      12.7 74                   Propionic acid peak                                                                            44   10.5       613.sup.b                                                                              58.3      86.4 174                  __________________________________________________________________________     .sup.a % binding to .sup.125 IYD.sub.4 K at 1:1,000 dilution ×          volume (ml)                                                                   .sup.b difference in cpm in presence of 10.sup.- 5 M D.sub.4 K and absenc     of D.sub.4 K binding to immobilized peptide at 1:100 dilution; detection      by .sup.125 Igoat antirabbit × volume (ml)                         

SPECIFICITY OF ANTIBODIES FOR C-TERMINAL D₄ K PEPTIDES

The relative affinities of affinity-purified Ca²⁺ -dependent antibodiespresent in sera from rabbit 1 for various peptides was examined bydetermining competition of binding to ¹²⁵ I-YD₄ K, measured byprecipitation with a second antibody, donkey anti-rabbit. Syntheticpeptides D₄ K, VD₄ K and APFD₄ K, displaced binding at similarconcentrations (FIG. 3) with IC₅₀ values all in the region of 10⁻⁸ M. Incontrast Asp, Lys, Asp-Lys, Asp-Glu, poly-Asp, and human gastrin(residues 1 to 17 containing pentaGlu sequence) did not displace bindingto ¹²⁵ I-YD₄ K at concentrations as high as 10⁻⁴ M (FIG. 3). Somebinding to Asp₂ Lys was seen. Affinity purified Ca²⁺ -dependent and Ca²⁺-independent antibodies from other rabbits showed a very similarspecific affinity and specificity for tetra-L-aspartyl-L-lysinesequences.

Of great importance to the general principle of the PAP assay of whichthis is an example, and for the application of these specific anti-D₄ Kantibodies to the diagnosis of pancreatic disease was the finding thatanti-D₄ K antibodies were C-terminally directed on the peptide, and werenot displaced by trypsinogen (where the peptide is C-terminally bound tothe protein) in concentrations of the zymogen of up to 100 μg/ml. Uponincubation of trypsinogen with enteropeptidase, however, released D₄K-containing activation peptides actively displaced the binding ofsynthetic ¹²⁵ I-YD₄ K giving 50% displacement after activation of a 2μg/ml solution of trypsinogen (FIG. 4). Trypsinogen in native dogpancreatic juice was also activated by enteropeptidase (FIG. 4)releasing immunoreactive D₄ K peptides, giving 50% displacement incompetitive solution-phase immunoassay after activation of 10⁻⁸ Lpancreatic juice. The novel D₄ K-specific antibodies described heretherefore also provide a new sensitive assay for enteropeptidase andtrypsinogen applicable to fluids with endogenous trypsin inhibitors.

The singular specificity for D₄ K peptides of the novel anti-D₄ Kantibodies we produced, was further demonstrated by examining skinsecretions of the amphibian Xenopus laevis. Tetra-aspartyl-lysylpeptides as internal sequences in larger proteins, had been predicted tooccur in the skin of this species by finding the corresponding nucleicacid sequence in cDNA clones derived from cutaneous mRNA [53]. Skinsecretions from this amphibian are known to contain a variety ofpeptides with counterparts in the mammalian nervous system includingCCK, gastrin, enkephalin, TRH, and somatostatin. We obtained a crudepeptide extract derived from the skin of this species and subjected itto reverse-phase HPLC. on a C8 column. The elution profile demonstratingmultiple peptides is shown in FIG. 5a. Only two peaks immunoreactive forD₄ K peptides were, however, identified (FIG. 5b) and theseco-chromatographed with the migration positions identified in thissystem for synthetic D₄ K and APFD₄ K. Free D₄ K C-termini were thoughtto have been generated in the crude peptide mixture by processing ofprecursors, although the titre of available D₄ K C-termini was increasedby tryptic hydrolysis of the peptide extract indicating the presence ofsome unprocessed precursor. D₄ K sequences in frog skin are thought toarise as a result of distant evolutionary relationships to mammaliantrypsinogens. The specific recognition of D₄ K peptide in the crudemixture obtained from this amphibian, further demonstrates thespecificity of the affinity-purified anti-D₄ K antibody.

IMMUNOASSAY OF D₄ K PEPTIDES Solution Phase CompetitiveImmunoradiometric Assay

100 μl of a 1:250 dilution of anti-D₄ K antiserum or a dilution ofaffinity-purified anti-D₄ K antibodies in 50mM Tris-Hcl, 20mM CaCl₂,0.1% (w/v) BSA, pH 7.4 (RIA buffer) were added to 100 μl of ¹²⁵ I-YD₄ K,(10,000 cpm) in RIA buffer containing 0.2% (v/v) normal rabbit serum inpolystyrene tubes. Then 100 μl of solutions containing variousconcentrations of standard peptides or unknown plasma samples diluted inRIA-buffer were added, followed by 50 μl of donkey anti-rabbit Ig serum(Wellcome Biotechnology) diluted 1:10 with RIA-buffer with 0.2% normalrabbit serum. The mixtures were incubated 18 hours at 4° C., then thetubes were centrifuged at 3,000 rpm for 45 minutes at 4° C., and thesupernatants aspirated. The radioactivity in the precipitates wasdetermined in an LKB rack-gamma counter.

Solid-phase Competitive Immunoradiometric Assay

In order to improve the ease and sensitivity of immunoassay over thatassociated with competitive precipitation radioimmunoassay, two types ofassay were developed using solid-phase techniques. In the first type,wells of PVC. microtitre plates were first coated with BSA-YD₄ K at 60μg/ml protein concentration. Then standard concentrations of syntheticactivation peptides were preincubated with affinity-purified anti-D₄ Kantibodies and applied to the wells. Antibody adhering to theimmobilised peptide hapten was then determined with ¹²⁵ I-labelled goatanti-rabbit antibody.

A typical standard curve is shown in FIG. 6, using Ca²⁺ -independentantibody. A steep dependence on the concentration of competing peptidewas apparent over the range 10⁻⁶ M to 10⁻⁹ M with IC₅₀ value of 5×10⁻⁷.

A second type of radiometric assay was developed using immobilisedaffinity-purified anti-D₄ K antibody. Wells of microtitre plates werecoated with 50 μl of Ca²⁺ independent antibodies (50 μg/ml) at 4° C.overnight, in 50mM Tris-HCl (pH 7.4) with 0.1% (w/v) sodium azide.Plates were then washed three times with 50mM Tris-HCl (pH 7.4)containing 10% horse-serum (T-HS buffer), and incubated with T-HS bufferfor 1 hour at room temperature to block remaining sites. Solutions (60μl) were prepared containing ¹²⁵ I-YD₄ K (100,000 cpm) and variousconcentrations of D₄ K (10⁻⁴ M -10⁻¹³ M), in T-HS buffer. 50 μl aliquotswere transferred to the antibody-coated wells, then incubated at roomtemperature for 5 hours. Plates were washed with T-HS buffer threetimes, and left to dry at room temperature. The wells were cut out andradioactivity determined as described. The results (FIG. 7) showed awider dependence on cpm bound between 10⁻⁶ M and 10⁻¹² M of competingpeptide, and an IC₅₀ value of about 10⁻⁹ M representing a 10-foldincrease in sensitivity.

Solid-phase enzyme-linked immunoassay

Benzidine dihydrochloride (10.2 mg) in 0.2M HCl (2 ml) was diazotised byaddition of sodium nitrite (7.8 mg) in water (0.2 ml) for 1 hour at 4°C., then buffered to pH 9 with 3.2 ml of 0.25M sodium borate/0.2M NaCl.Then YD₄ K (4.9 mg) was coupled to horseradish peroxidase (Sigma; 49.2rag) in 10.8 ml 0.16M sodium borate/0.14M NaCl (pH 9) by addition of thebenzidine reagent at +4° C., and incubating for 2 hours. Excess reagentsand peptide were removed by extensive dialysis against 0.15M NaCl,water, then 0.15M NaCl to yield a solution of conjugate of 3.2 mg/ml.

ELISA assay was performed as described above for the second type ofradiometric assay using plates coated with Ca²⁺ independent antibody (25μg/ml) in 50mM Tris (pH 7.4), 0.1% sodium azide. Plates were washedthree times with 50 mM Tris (pH 7.4), 10% (v/v) horse serum, 0.5% Tween20, blocked with 50mM Tris (pH 7.4), 10% (v/v) horse serum for 30minutes, then incubated with a mixture of 1:10,000 peroxidase-D₄ Kpeptide conjugate and unknown samples or standard amounts of peptide in50mM Tris (pH 7.4), 0.05% Tween 20. After 1 hour at 20° C., the plateswere washed three times and developed by incubation with3,3',5,5'-tetramethylenebenzidine (0.01% [-w/v] in 0.1M sodiumacetate/citric acid [pH 6.6] for 1 hour at 20° C. The reaction wasstopped by addition of 50 ml 2M H₂ SO₄, and colour estimated at 450nm.The resulting displacement curve exhibited similar sensitivity to theradiometric assay shown in FIG. 7.

ASSAY OF THE STABILITY AND DISTRIBUTION OF D₄ K PEPTIDES IN VIVO

The D₄ K peptide was found to be stable in human serum, and activatedpancreatic juice. Immunoreactive D₄ K was unaltered by incubation withundenatured human serum at 37° C. for 6 hours and 4° C. for 48 hours.Sephadex G-100 chromatography of YD₄ K after incubation in human serumshowed that the immunoreactive D₄ K peptide co-chromatographed with freepeptide in buffer and did not complex with a serum component.

The stability of APFD₄ K and D₄ K were examined by incubation withactivated pancreatic juice, and the chemical identity of the productexamined by high-voltage paper electrophoresis at pH 6.5. The resultsshowed that APFD₄ K was rapidly converted to free Ala, Pro and Phe andD₄ K, but that the D₄ K sequence itself was stable for 24 hours at 37°C. In three separate experiments, duplicate 65 to 75 mg portions offresh dog pancreas were placed in modified Bank's medium and incubatedin the presence or absence of 100 units enterokinase (Sigma Chem. Co.E0885) at 37° C. for 4 hours and 22° C. for a further 20 hours.Duplicate 100 ul samples of culture medium supernatant were taken atintervals, diluted 2:1 with RIA buffer, boiled for 10 minutes andcentrifuged. Supernatants were then stored at -20° C. prior to assay forD₄ K peptides.

In each of the three experiments incubation with enterokinase wasassociated with degradation of pancreatic tissue and rapid release toimmunoreactive D₄ K peptides. These changes were not seen in short termcultures without enterokinase (FIG. 8). D₄ K immunoreactive peptides inthe culture medium persisted at high levels after 24 hours incubationdemonstrating their resistance to combined pancreatic proteolysis.

To determine the fate of circulating peptide, D₄ K (0.28 ml, 1 mg/ml)was injected into the left femoral vein of rats under phenobarbitalanaesthesia. Blood samples (0.5 ml) were collected every 15 minutes for2 hours, and subjected to immunoassay. The results showed thatadministered immunoreactive D₄ K disappeared from serum in vivo withhalf-life of about 15 minutes. In a second series of such experimentsYD₄ K 100 μg/kg in 1 ml PBS was injected intravenously in each of threehealthy fasted greyhound dogs. Samples of blood and urine were obtainedat intervals over 4 hours and stored at -20° C. prior to assay. The meanhalf-life of D₄ K peptide in blood was 8.3 minutes in dogs but in eachcase D₄ K was detectable in serum for 2 hours. D₄ K appeared in urinewithin 5 minutes of intravenous administration and persisted longer, for3 to 4 hours (FIG. 9).

A limited study was carried out to determine the tissue distribution ofimmunoreactive D₄ K peptides. Extracts of rat brain, pancreas, duodenumand pituitary were prepared by homogenisation and sonication in 24mM HClcontaining pepstatin 0.1 mg/ml, chymostatin 0.1 mg/ml, elastinal 0.1mg/ml, and Trasylol 2000 KN/ml. Extracts were adjusted to pH 5.6,diluted with 0.1M succinate buffer pH 5.6, and digested withenteropeptidase for 1 hour at 37° C. 6×10⁻⁷ moles D₄ K/mg protein and1.5×10⁻⁷ moles D₄ K/mg protein were found after enteropeptidasedigestion in extracts of rat pancreas and duodenum respectively. No D₄ Kpeptide was detected in extracts of rat brain or pituitary, and none wasfound after assay of extracts of guinea-pig brain similarly prepared.Canine pancreatic juice obtained by cannulation of the main pancreaticduct followed by CCK stimulation, showed no immunoreactive D₄ K peptidebefore enteropeptidase activation and 5.7×10⁻⁴ moles D₄ K/ml afteractivation. No D₄ K immunoreactivity was identified in fasting orpostprandial serum or urine in multiple samples from 3 dogs and 6 normalhuman subjects. No D₄ K immunoreactivity was found in serum samples ofpatients with perforated duodenal ulcer, mesenteric infarction,appendicitis, aortic aneurysm, Crohn's disease or gastrointestinalhaemorrhage. No D₄ K was found in human sera in association withAltzheimer's disease, rheumatoid arthritis or any other non-pancreaticdisease state tested.

ASSAY OF D₄ K PEPTIDE IN EXPERIMENTAL AND HUMAN PANCREATITIS

Acute necrotising pancreatitis was induced experimentally in 22anaesthetised rats by the controlled intrapancreatic duct microinfusionover 30 minutes of 75 ul buffer containing 10-20 mmol/1glycodeoxycholate alone or with 50ng highly-purified humanenteropeptidase [54]. The rats subsequently received continuousintravenous analgesia together with pancreatic stimulation using CCK-3310 IDU/kg/h. Blood was taken before the intraduct infusion and 3 hoursafterwards, and assayed for free D₄ K peptide. Histological examinationof the pancreas showed severe acute pancreatitis in every case.Circulating immunoreactive D₄ K peptide rose with the development of thedisease in each of the 22 animals from an apparent mean basal level of 5pmole/ml to a mean of 84 pmole/ml after 3 hours. By contrast no free D₄K peptides were identified in pancreas homogenates from 6 mice withacute oedematous pancreatitis induced by CCK-8 hyperstimulation [55].

Preliminary clinical studies were carried out to determine whether D₄ Kpeptide could be identified in serum or urine from severe acutepancreatitis patients as predicted. Random serum and in some cases urinesamples were accumulated from 22 patients with pancreatitis. Not all ofthese samples were taken during the early hours of the disease. On thebasis of Ranson's criteria [56] 14 patients were classified as havingthe milder oedematous form of pancreatitis and 8 patients were assessedas having the severe necrotising form of the disease. No immunoreactiveD₄ K peptide was found in samples from any of the patients with theoedematous disease despite substantial elevation of the serum amylase inall of them, whereas D₄ K was identified in samples of serum and/orurine from 6 of the 8 judged to have severe pancreatitis (Table 2).

                  TABLE 2                                                         ______________________________________                                                                D.sub.4 K                                                    Aetiology       Pancreatitis     Se-                                   Initial                                                                             Age    GS     Etoh Other Mild Severe                                                                              Urine rum                           ______________________________________                                        M.B.  76     +                      +     +                                   H.B.  50                 +     +                                              J.B.  84     +                 +                                              D.D.  53     +                      +           +                             D.G.  70     +                 +                                              R.G.  66     +                      +                                         W.G.  56                 +          +           +                             A.J.  38            +          +                                              D.K.  84                 +     +                                              J.K.  33            +          +                                              P.L.  67                 +          +                                         G.M.  41                 +     +                                              T.N.  63                 +     +                                              V.N.  30            +          +                                              I.N.  53     +                 +                                              T.P.  43            +          +                                              J.P.  68     +                 +                                              M.R.  36            +          +                                              A.R.  81     +                 +                                              C.R.  54     +                      +     +     +                             O.S.  48                 +          +           +                             J.S.  45                 +          +           +                             ______________________________________                                         TAP Assay of random samples collected at St. George's January 1985-March      1987. Stored at -20° C.                                           

Chromatography of samples of JS sera on Sepharose G15 showed that theimmunoreactivity co-migrated with standard APFD₄ K (FIG. 10). Inaddition, serum was obtained from one patient (PD) who presented withabdominal pain and an exacerbation of chronic pancreatitis for which hehad previously had a 75% distal pancreatic resection. D₄ K peptide wasdetected in this sample at the level of 10⁻⁸ M.

In a second clinical study assay for D₄ K peptides was carried out onsera accumulated during routine daily sampling from 69 patients admittedto the Glasgow Royal Infirmary, Scotland, and other hospitals in theGlasgow area (FIG. 11). Sera were stored frozen at -20° C. and had to bethawed, aliquoted, and refrozen prior to rethawing and assay. A total of279 stored sera were available representing serum samples taken from the69 patients for up to 5 days, but in a few cases only a single samplewas available. Assay for D₄ K peptides was performed blind withoutknowledge of the severity or clinical course of the pancreatitis. 36 of39 Patients (92%) assessed as having oedematous pancreatitis were D₄ Knegative and 13 of 23 more severe cases (56%) were D₄ K positive. Theoverall accuracy of the D₄ K assay performed retrospectively on routinefrozen-thawed-frozen-thawed serum-only samples in this series was 79%.Apparent false positives (3) probably reflect the known inadequacy ofthe clinical assessment method. Apparent false negatives (10) are likelyto reflect this, together with errors introduced by the dissociation ofroutine once a day serum sampling from changing clinical status,and theabsence of the additional information contributed by a urinary assay.

Three patients admitted to St. George's Hospital, London, SW17, England,with acute pancreatitis and hyperamylasaemia were studied using the D₄ Kassay as intended, to monitor the severity and progress of the disease.To do this, simultaneous blood and (where available) urine samples weretaken immediately on admission and at 4 to 6 hourly intervals for thefirst 48 hours and thereafter 8 hourly. Samples were stored frozen at-20° C. prior to D₄ K assay. One patient TP (FIG. 12a) withmoderate/severe pancreatitis but who later developed a pseudocyst, hadan initial urinary D₄ K level of 250 pmol ml⁻¹ 24 hours after admissionbut no D₄ K detectable in serum at this time. This reflects thelook-back capability of the urine assay and the prolonged urinaryexcretion of D₄ K peptide after intravenous administration identifiedexperimentally in dogs. On the fourth day of hospitalisation, D₄ Kpeptides appeared transiently in serum and later rose in urine. Thesechanges were matched by a distinct clinical exacerbation supporting therole of the D₄ K and other PAP assays as a precise disease-coursemonitor.

In a second patient CM (FIG. 12b) in this series with moderatepancreatitis but with respiratory insufficiency, serum D₄ K with 38 pmolml⁻¹ and urinary D₄ K 100 pmol ml⁻¹ on admission Both fell to zerowithin a few hours and thereafter remained negative, but a 2 cmpseudocyst and small lesser sac fluid collection was subsequentlyidentified. This further supports the ability of D₄ K assay to predicteven small areas of pancreatic necrosis. The third patient in thisgroup, CTM, (FIG. 12c) admitted with abdominal pain and a serumamylase >7000 units, remained serum and urine D₄ K negative over 5 daysdespite his initial signs. He had gall stones with oedematouspancreatitis which subsequently resolved over 5 days. This againsupports the role of a negative D₄ K assay as an accurate severityindicator in the acute disease. These results also suggest that thesignificant number of false negative D₄ K assays in the Glasgowserum-only study would not have occurred with frequent blood and urinesampling and the assay applied clinically as intended.

Additional serum samples from two patients with chronic recurrentpancreatitis in exacerbation showed D₄ K peptides but serum amylaselevels within normal limits; this would support the diagnosticcapability of the D₄ K assay in the chronic disease. Taken together theclinical and experimental findings with the D₄ K assay provide strongsupport for the proposed role of the invention in the diagnosis,severity prediction, and disease-course-monitoring of pancreatitis. Thefindings also support the principle embodied in the invention for thespecific assay of body fluids for free PAP molecules using C-terminallydirected antibody in pancreatic diseases.

PAP assays will differentiate different forms of pancreatic disease asfollows:

TAP assays specifically report pathological trypsinogen activation. TAPassay will be positive in body fluids including blood and urine in casesof necrotising pancreatitis and negative in body fluids in cases ofoedematous pancreatitis. Since the release of other PAP including PLAP,CLAP and PEAP will occur as a second generation of activation eventsfollowing trypsinogen activation in necrotising acute pancreatitis, freePLAP, CLAP, PEAP and other PAP will also be present in body fluids inthis condition. Differences in the rates of release and elimination ofvarious PAP including TAP, PLAP, CLAP, and PEAP will permit theidentification of disease sub-groups within necrotising acutepancreatitis. Since the specific release of PAP is equimolar with theproduction of the corresponding active enzyme a quantitativerelationship will exist between PAP levels in body fluids and diseaseseverity enabling PAP assays to serve as early precise predictors ofseverity and necrosis in acute pancreatitis. Since pancreatic zymogenactivation is also involved in chronic pancreatitis but not generally inpancreatic cancer or other abdominal diseases such as peptic ulcer orchronic cholecystitis, positive PAP assays in body fluids willdistinguish diagnostically between chronic pancreatitis in exacerbationand other causes of chronic or intermittent abdominal pain.

OTHER PAP ASSAYS OF THE INVENTION

Activation peptides of other pancreatic zymogens, and their counterpartssharing activation peptide C-terminal sequence homology occurring inother cells and tissues, which are suitable for specific assay usingC-terminally directed antibodies for the free species in accordance withthe present invention, include those of prophospholipase A₂,procolipase, proelastase 1 and proelastase 2, prekallekreins and ofprocarboxypeptidases A & B. Examples of these are peptides containingthe carboxy terminal sequence Asp-Ser-Gly-Ile-Ser-Pro-Arg ofprophospholipase A₂ [41], peptides containing carboxy terminalAla-Pro-Gly-Pro-Arg of procolipase [42 ], and peptides containingcarboxy terminal Gly-Asp-Pro-Thr-Tyr-Pro-Pro-Tyr-Val-Thr-Arg ofproelastase 2 [43] or peptides representing the sequence of theactivation peptides of proelastase 1 or prekallekreins. Portions ofthese sequences representing proteolytic degradation oligopeptides of 5or more residues such as Pro-Pro-Tyr-Val-Thr-Arg from proelastase 2 mayalso be used. The much larger activation peptides ofprocarboxypeptidases A & B may also contain oligopeptide sequencessuitable as targets. The use of C-terminally directed antibody willensure that zymogen forms normally present in human plasma and urine[57] will not be recognised in the assay as in the segregation of D₄ Kcontaining free peptides from trypsinogens, already given as a detailedexample.

Of additional interest and importance is the activation peptide ofprophospholipase A (PLAP). Prophospholipases A are abundant in many celltypes including macrophages [58] pulmonary alveolar macrophages [59],aggregated platelets, and polymorphnuclear leucocytes [61] as well asbeing generally present in lysosomes. Active phospholipase A₂ isreleased from these in several inflammatory conditions [62] includingendotoxin shock 63], respiratory distress syndrome, acute abdominalconditions and in response to mycobacteria [61], peptides [60] and Ca²⁺[39]. If the prophospholipase A₂ from these sources is the same geneproduct as pancreatic prophospholipase A₂ and has an activation peptideof the same sequence Asp-Ser-Gly-Ile-Ser-Pro-Arg, PLAP assay as definedin this invention, will also be a useful test applicable to therecognition and severity prediction of these disorders. If thenon-pancreatic prophospholipase A₂ activation peptide has a sequencedifferent from pancreatic prophospholipase A₂, then the PLAP assay willbe pancreatic disease specific.

PLAP assays in body fluids will be applicable diagnostically and asdisease activity indicators in chronic intermittent inflamatoryconditions such as rheumatoid arthritis, Crohn's disease anddermatomyositis, in demyelinating neurological disorders, and in acuteconditions such as endotoxic shock, respiratory distress syndrome, andthe acute abdomen.

The involvement of a procolipase (sharing sequence homology in theactivation peptide C-terminal region with the pancreatic procofactor) inlipolytic activity in other tissues including liver, adipocytes andskeletal muscle, will implicate CLAP assays diagnostically and asdeterminants of disease status where disorders of lipid metabolism areinvolved in the molecular pathology of these diseases. These includeinherited hyperlipidaemias, diabetes mellitus, alcoholic liver diseaseand morbid obesity but disorders such as anorexia nervosa may also berelevant.

PEAP assays reporting activation of proelastases showing sequencehomology in the activation peptide C-terminal region with pancreaticisoenzymes, are implicated diagnostically and as activity determinantsin diseases where elastase degradation is involved in disease process.Such diseases include osteophorosis, pulmonary emphysema and arterialdegeneration.

Synthesis of prophospholipase A₂ activation peptide (PLAP) andprocolipase activation peptide (CLAP)

Further examples of the invention involve the synthesis by solid-phasemethods, of peptides relevant to the development of immunoassaysC-terminally specific for PLAP and CLAP. These peptides areAsp-Ser-Gly-Ile-Ser-Pro-Arg, Cys-Tyr-Asp-Ser-Gly-Ile-Ser-Pro-Arg,Ala-Pro-Gly-Pro-Arg and Cys-Tyr-Ala-Pro-Gly-Pro-Arg. Syntheses wereperformed on p-alkoxybenzyl-alcohol-derivatised polystyrene (0.67 mequivs/g from Bachem Feinchemikalien AG, Switzerland) using temporatorybase-labile Fmoc/alpha-amino group protection and the general methodsdescribed by Cliffe et al [50] with the following modifications. TheC-terminal residue was coupled as its protected derivative Fmoc (Mtr)Argby a mixed anhydride method. Two equivalents of protected Arg wereincubated with resin having one equivalent of alkoxybenzyl group,together with two equivalents of 2,6-dichlorobenzoyl chloride and threeof redistilled pyridine in dimethylformamide for 16 hours. Allsubsequent Fmoc amino acids were coupled in a 2-fold excess as1-hydroxybenzotriazole esters. The following derivatives were employed:Fmoc-Pro, Fmoc-(0tBu)Ser, Fmoc-Ile, Fmoc-Gly, Fmoc-(OtBu)Asp,Fmoc-(0tBu)Tyr and Fmoc-(SAcM)Cys. At the end of the synthesis, theN-terminal Fmoc group was cleaved by treatment with 20% (v/v) piperidinefor 10 minutes and the peptide cleaved and partly deprotected bytreatment with 50% (v/v) trifluoroacetic acid, 5% thioanisole, 45% (v/v)dichloromethane. This was evaporated, washed four times with ether andlyophilised. Portions (250 rag) of those peptides containing protected(SAcM) cysteinyl groups were extracted into minimal volumes of water andadjusted to pH4 with NH₄ OH. Then mercuric acetate (200 rag) was addedand the pH readjusted to 4. After 1 hour, the peptide was diluted to 50ml and H₂ S passed through the solution for 15 minutes, followed by N₂for 10 minutes. The black precipitate (HgS) was separated bycentrifugation and the peptide recovered by lyophilisation. Peptideswere purified by gel chromatography on Sephadex G-15 in 0.1M acetic acidand characterised by reverse phase HPLC. and amino acid analysis. OnBrownlee C-8 Aquapore RP-300 in 0.05% trifluoroacetic acid andacetonitrile from 5% to 25% (v/v) over 22 minutes at 1.0 ml/min peptidesgave single peaks at 9.14 minutes for CYAPGPR, 5.49 minutes for APGPR.On uBondapak C₁₈ in 0.05% trifluoroacetic acid running and acetonitrilegradient 5% to 50% over 30 minutes 1.5 ml/min CYDSGISPR eluted at 11.7minutes and DSGISPR at 7.8 minutes. Amino acid analysis confirmed thecorrect composition of these peptides.

Peptides with Cys-Tyr amino terminal extensions are used forhaptenisation or the attachment of revealing agent or solid phase, andnative peptides for use on solid phase or as competing ligands asdescribed in the D₄ K example of the invention. Specific C-terminalantibodies either polyclonal or monoclonal are purified by affinitychromatography on the corresponding immobilised peptide and used in theperformance of solid or solution phase assays as detailed in the D₄ Kexample. Peptides with revealing agents or immobilised on solid phase aswell as specific antipeptide antibodies with or without revealing agent,and free or attached to solid phase, and the use of such reagents in theperformance of PAP assays in the diagnosis and severity monitoring ofdisease form further aspects of the invention.

BIBLIOGRAPHY

1. Corfield A. P. et al (1985, Gut 26:724-729.

2. Hermon-Taylor, J. and Heywood, G. C. (1985), Scand. J. Gastroenterol.20 (Suppl. 117), 39-46.

3. Salt, W. B. and Schenker, S. (1976). Medicine 55:269-289.

4. Temler, R. S. and Felber, J-P. (1976) . Biochim. Biophys. Acta. 445:720-728.

5. Elias, E. et al (1977), Lancet ii:66-68.

6. Borgstrom, A. and Ohlsson, K. (1978), Hoppe-Seyler's Z. Physiol.Chem. 359:677-681.

7. Adrian, T. E. et al, (1979), Clin. Chim. Acta. 97:205-212.

8. Lake-Bakaar, G. et al, (1979), Lancet ii:878-880.

9. Crossley, J. R. et al, (1979), Lancet i:472-274.

10. Brodrick, J. W., et al, (1979), Am. J. Physiol. 237:E474-E480.

11. Geokas, M. C. et al, (1979), Am. J. Physiol. 236: E77-E83.

12. Koop, H. et al, (1980) , Digestion 20: 151-156.

13. Duffy, M. J. et al, (1980), Clin. Chim. Acta, 103:233-235.

14. Malvano, R. et al, (1980), Scand. J. Gastroenterol, 15 (Suppl.62):3-62.

15. Geokas, M. C. et al, (1981), Am. J. Pathol. 105:31-39.

16. Farini, R. et al, (1981), Gastroenterology 81:242-246.

17. Masoero, G. et al, (1982), Dig. Dis. Sci. 27: 1089-1094.

18. Steinberg, W. M. and Anderson, K. K. (1984), Dig. Dis. Sci, 29:988-993.

19. Umeki, S, et al, (1985), J. Lab. Clin. Med. 106:578-582.

20. Wellborn, J. C. et al, (1983), Am. J. Surg. 146:834-837.

21. Geokas, M. C. et al, (1979), J. Biol. Chem. 254:2775-2781.

22. Eskola, J. U. et al, (1983), Clin. Chem. 29:1777-1780.

23. Nevalainen, T. J. et al, (1985) , Clin. Chem. 31:1116-1120.

24. Delk, A. S. et al, (1985), Clin. Chem. 31:1294-1300.

25. Goldberg, J. M. (1976), Clin. Chem. 22:638-642.

26. Mayer, A. D. et al, (1985), Br. J. Surg. 72:436-437.

27. Kitahara, T. et al, (1980), Clin. Chim. Acta. 103:135-143.

28. Otsuki, M. et al, (1984), Clin. Chim. Acta. 142: 231-240.

29. O'Connor, C. M. et al, (1981), Clin. Chim. Acta. 114:29-35.

30. Hermon-Taylor, J. et al, (1981) , Clin. Chim. Acta. 109: 203-209.

31. Massey, T. H. (1985), Clin. Chem. 31:70-75.

32. Pace, B. W. et al, (1985), Am. J. Gastroenterol. 80:898-901.

33. Mifflin, T. E. et al, (1985), Clin. Chem. 31:1283-1288.

34. Moller-Petersen, J. et al, (1986), Clin. Chim. Acta. 157:151-166.

35. DiMagno, E. P. (1983), Alabama J. Med. Sci. 20:404-410.

36. Spechler, S. J. et al, (1983), Dig. Dis. Sci. 28:865-869.

37. Eckfeldt, J. H. et al, (1985), Arch. Pathol. Lab. Med. 109:316-319.

38. Puolakkainen, P. et al, (1987), Gut. 28:764-771.

39. Traynor, J. R. et al, (1981), Biochim. Biophys. Acta. 665:571-577.

40. Vadas, P. (1984), J. Lab. Clin. Med. 104:873-881.

41. Grataroli, R. et al, (1982), Eur. J. Biochem, 122:111-117.

42. Sternby, B. et al, (1984), Biochim. Biophys. Acta. 786:109-112.

43. Largman, C. et al, (1980), Biochim. Biophys. Acta. 623:208-212.

44. Maroux, S. et al, (1971), J. Biol. Chem. 246:5031-5039.

45. Broderick, J. W. et al, (1978), J. Biol. Chem. 253:2737-2742.

46. Stanley, C. J. et al, (1985), J. Immunol. Methods 83:89-95.

47. Carpino, L. A. and Han, G. Y., (1972), J. Org. Chem. 37:3404-3409.

48. Wang, S. (1973), J. Amer. Chem. Soc. 95:1328-1333.

49. Meienhofer, J. et al, (1979), Int. J. Pep. Prot. Res. 13:35-42.

50. Cliffe, S. G. R. et al, (1985), Int. J. Pep. Prot.Res. 25:663-672.

51. Bassiri, R. M. and Utiger, R. D. (1972), Endocrinology, 90:722-727.

52. Green, N. et al, (1982), Cell 28:477-484.

53. Hoffman, W. et al, EMBO. J. (1983), 2:711-714.

54. Cawthorne, S. J. et al, (1984), Dig. Dis. Sci. 29:945.

55. Shorrock, K. et al, (1986), Gut. 27:A1260.

56. Ranson, J. H. C. et al, (1974), S. G. O. 139:69-81.

57. Sternby, B. et al, (1984), Biochim. Biophys. Acta, 789:164-69.

58. Vadas, P. and Hay, J. B. , (1980), Life Sci. 26:1721-29.

59. Lanni, C. et al, (1981), Biochim. Biophys, Act. 658:54-63.

60. Lanni, C. et al, (1983), Am. J. Pathol. 113:90-94.

61. Franson, R. C. et al, (1973), J. Cell. Biol. 56:621-627.

62. Franson, R. C. et al, (1978), J. Lipid. Res. 19:18-23.

63. Vadas, P. (1984), J. Lab. Clin. Med. 104: 873-881.

We claim:
 1. A immunological method of detecting the activation ofpancreatic zymogens in a patient which comprises:(a) providing a sampleof the patient's body fluid; and (b) detecting the presence or absencein the sample of peptides which have the same carboxy-terminalpentapeptide sequence as the activation peptides of pancreatic zymogens(PAP), the pancreatic zymogen being selected from the group consistingof trypsinogen, prophospholipase A₂, procolipase, proelastase 1,proelastase
 2. prekallekrein, procarboxypeptidases A andprocarboxypeptidases B, said presence of absence indicating theexistence or progress of the activation of said zymogens in saidpatient.
 2. A method according to claim 1 for diagnosing or monitoringthe progress of pancreatic disease.
 3. A method according to claim 2wherein the peptide assayed for is a trysinogen activation peptide (TAP)comprising the amino acid sequence tetra-L-aspartyl-L-lysine (D₄ K)having the lysine (K) as the carboxy terminus.
 4. A method according toclaim 1 wherein the peptide assayed for is human prophospholipase Aactivation peptide (PLAP) comprising the amino acid sequence

    D S G I S P R

having the R as the carboxy terminus or procolipase activation peptide(CLAP) comprising the amino acid sequence

    A P G P R

having the R as the carboxy terminus or proelastase 2 activation peptide(PEAP) comprising the amino acid sequence

    G D P T Y P P Y V T R

having the R as the carboxy terminus or the degradation product ofproelastase 2 activation peptide (PEAP) comprising the amino acidsequence

    P P Y V T R

having the R as the carboxy terminus.
 5. A method according to claim 4for the diagnosis of non-pancreatic disease which comprises assaying asample of the patient's body fluid for the presence or absence ofpeptides which are PLAP, CLAP or PEAP including the activation peptideof proelastase
 1. 6. A method according to claim 1 wherein the bodyfluid is blood, serum, ascites or urine, or cerebrospinal fluid.
 7. Amethod according to claim 1 wherein the PAP is trypsinogen activationpeptide.
 8. A method according to claim 1 wherein said assaying is doneas a solid/liquid phase reaction involving the formation of a conjugatebetween the PAP and a specific antigen binding site of a C-terminallydirected antibody which specifically binds PAP, said conjugate carryinga revealing label and being formed either in the solid phase or in theliquid phase of the solid/liquid phase reaction mixture, wherein saidmethod further comprises separating the solid phase from the liquidphase and determining the presence of or amount of the revealing labelin either the solid phase or the liquid phase as a measure of thepresence of or amount of, respectively, PAP in the sample.
 9. A methodaccording to claim 1 wherein said assaying is done in a homogeneousassay system involving the formation of a conjugate between PAP and aspecific antigen binding site of a C-terminally directed antibody whichspecifically binds PAP and said conjugate is formed in the liquid phaseof the homogeneous assay system.
 10. A method according to claim 8carried out as an ELISA using antibody labelled with enzyme or biotin.11. A method according to claim 8 carried out as an ELISA using PAPlabelled with enzyme or biotin.
 12. A method according to claim 1wherein the sample is brought into contact with a solid phase comprisingan inert solid support to which is bound either (1) PAP having bound toit by the specific antigen binding site an antibody to PAP, the antibodycarrying a revealing label, or (2) antibody to PAP having bound to it bythe specific antigen binding site PAP, the PAP carrying a revealinglabel.
 13. A method according to claim 1 wherein samples of body fluidare removed from the patient on at least two separate occasions spacedapart from one another by at least one half hour and each sample isassayed for the concentration of peptides which have the samecarboxy-terminal pentapeptide sequence as PAP as a means for monitoringthe severity or progress of pancreatitis.
 14. A solid component for adiagnostic test kit comprising the trysinogen activation peptide D₄ K ora hapten conjugate thereof, said D₄ K or hapten conjugate immobilised onan inert solid support having the lysine (K) as a free carboxy terminus,said D₄ K or D₄ K hapten conjugate further bound to a C-terminallydirected antibody which specifically binds an activation peptide of apancreatic zymogen (PAP).
 15. A solid component for a diagnostic testkit comprising an activation peptide of a pancreatic zymogen (PAP)selected from the group consisting of ##STR1## or a hapten conjugatethereof, said activation peptide or hapten conjugate immobilised on asolid support having the arginine (R) as a free carboxy terminus, saidpeptide or hapten conjugate further bound to a C-terminally directedantibody which specifically binds said activation peptide.
 16. The solidcomponent of claim 14 wherein said D₄ K or hapten conjugate furtherincludes a revealing label.
 17. The solid component of claim 15 whereinsaid peptide or hapten conjugate further includes a revealing label. 18.A solid component for a diagnostic test kit comprising a C-terminallydirected antibody which specifically binds an activation peptide of apancreatic zymogen (PAP), immobilised on an inert solid support to whichtrypsinogen activation peptide D₄ K, or a hapten conjugate thereof, isbound at its specific antigen binding site.
 19. A solid component for adiagnostic test kit comprising a C-terminally directed antibody whichspecifically binds an activation peptide of a pancreatic zymogen (PAP),immobilised on an inert solid support to the specific antigen bindingsite of which a peptide selected from the group consisting of DSGISPR,APGPR, GDPTYPPYVTR, and PPYVTR, or a hapten conjugate thereof, is bound.20. A diagnostic test kit comprising at least one component selectedfrom the group of activation peptides of a pancreatic zymogen (PAP)consisting of D₄ K, DSGISPR, APGPR, GDPTYPPYVTR, PPYVTR, or a haptenconjugate thereof, and a C-terminally directed antibody whichspecifically binds said activation peptide, said component carrying arevealing label.
 21. A diagnostic test kit of claim 20 wherein saidcomponent is immobilised on an inert solid support.
 22. A diagnostictest kit comprising a solid component and a liquid component, said solidcomponent selected from the group of activation peptides of a pancreaticzymogen (PA)P consisting of D₄ K, DSGISPR, APGPR, GDPTYPPYVTR, PPYVTR,or a hapten conjugate thereof, and a C-terminally directed antibodywhich specifically binds said activation peptide, said solid componentimmobilised on an inert solid support, and wherein one of saidcomponents carries a revealing label.